Importance of cytotoxic T lymphocytes in the rejection of transplanted hepatocellular carcinoma

Fischer rats became resistant to syngeneic hepatocellular carcinoma (FAA-HTC1) cells on repeated sensitization with mitomycin C-treated FAA-HTC1 cells. In contrast, FAA-HTC1 cells injected into the liver killed normal control Fischer rats within 2 months. Histopathological studies revealed massive accumulation of mononuclear cells in the tumor tissues of sensitized rats that rejected syngeneic FAA-HTC1 cells, whereas very few mononuclear cells were found in the tumor tissues of control rats. Cell populations infiltrating the tumor tissues were identified by flow cytometric analysis. Mononuclear cells found within the regressing tumors of the sensitized rats were identified as mostly T cells, and two-thirds of these T cells were CD8-positive. Compared with the activity in control rats, the killer activity of mononuclear cells infiltrating tumors was significantly increased in the sensititized rats 7 days after tumor inoculation. Depletion of CD8(+) T cells significantly reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from sensitized rats. In contrast, depletion of CD16(+) cells reduced the cytotoxicity of mononuclear cells infiltrating tumors obtained from both control and sensitized rats. Furthermore, the CD16(+) cell-depleted fraction of mononuclear cells infiltrating tumors showed significant cytotoxicity against FAA-HTC1 cells, but failed to show cytotoxicity against other syngeneic tumor cells or allogeneic hepatoma cells.     

Analysis of mononuclear cells infiltrating tumor in solitary hepatoma model

Ten million syngeneic hepatocellular carcinoma (FAA-HTCI) cells injected into liver are sufficient to kill Fischer rats within 2 months. Fischer rats became resistant to FAA-HTCI cells by repeated sensitization with MMC-treated FAA-HTC1 cells. Histopathological studies revealed massive accumulation of mononuclear cells in tumor tissues of sensitized rats that were rejecting syngeneic FAA-HTCI cells, whereas very few mononuclear cells were found in tumor tissues of control rats. Cell populations infiltrating the tumor tissues were identified by immunohistochemical techniques and flow cytometric analysis. Mononuclear cells found within the regressing tumors of the sensitized rats were mostly identified as being T cells, and two-thirds of these T cells were CD8 positive. Compared to control rats, killer activity of mononuclear cells infiltrating tumor tissues was significantly increased in the sensitized rats. Depletion of CD8(+) T cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating tumors obtained from sensitized rats. In contrast, depletion of CD 16(+) cells significantly reduced the cytotoxic ability of mononuclear cells infiltrating tumors obtained from control rats.     

Feedback and facilitation in the adrenocortical system: unmasking facilitation by partial inhibition of the glucocorticoid response to prior stress.

Previously stressed animals remain responsive to subsequent stressors, despite secreting an adequate corticosteroid signal during the first stress which should act to damp the response to a second stress. We have previously postulated that stress acts to facilitate subsequent responses in the adrenocortical system, and that this facilitation is balanced by the corticosteroid feedback signal. To test this hypothesis directly, we treated young male rats with cyanoketone (CK) to partially block the adrenal capacity to synthesize corticosterone (B). Subsequently, groups of CK- or vehicle (VEH)-treated rats were exposed to the FIRST stress of 30-min restraint with small blood samples collected at 0, 15, and 30 min. The FIRST stress was given to subgroups of rats 12, 9, 6, or 3 h before lights off (12 h) or lights on (24 h). At 12 or 24 h, rats were again restrained with blood samples at 0 ("basal") and 30 min (SECOND stress). Control groups were stressed for the first time when the experimental groups received their SECOND stress. Plasma ACTH and B concentrations were measured. Although in the absence of stress, basal B concentrations were normal in CK-treated compared to VEH-treated rats throughout the day, the B response to the FIRST stress was reduced by 60% in the CK- compared to the VEH-treated group. When the FIRST stress was performed during the time of lights on, "basal" plasma ACTH was elevated in CK groups at 12 h (lights off) compared to levels in both previously stressed VEH groups and unstressed CK controls. There was no difference at this time of day in the magnitude of the ACTH response to the SECOND stress in CK rats compared to that in CK rats receiving their only stress (controls) or that in VEH-treated rats receiving the SECOND stress. When first stress was performed during the time of lights off, "basal" plasma ACTH at 24 h (lights on) in CK and VEH rats were not different compared to levels in their respective unstressed controls. The ACTH response to the SECOND stress at 24 h was elevated in all previously stressed CK groups compared to that in either CK control or VEH groups. At neither time of day were SECOND stress ACTH concentrations in VEH rats different from those in control VEH rats. At 12 h (lights off), but not at 24 h (lights on), "basal" ACTH was significantly elevated in VEH rats above the unstressed VEH control values.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chronic stress increases experimental pancreatic cancer growth,reduces survival and can be antagonised by beta-adrenergic receptor blockade

Background/objectivesChronic stress could promote tumour growth and reduce survival of pancreatic cancer patients via beta-adrenergic receptors of tumour cells. We have tested the impact of chronic acoustic and restraint stress on tumour development in an orthotopic syngeneic murine model of pancreatic cancer.Methods and resultsTumour-bearing C57BL/6 mice exposed to chronic stress had 45% (p = 0.0138) higher circulating steroid and 111% (p = 0.0052) higher adrenal tyrosine hydroxylase levels. Their immune response was significantly suppressed: The in vitro LPS response of splenocytes was significantly reduced regarding Th1- and Th2-cytokines including IFN-gamma, IL-6, IL-10 and MCP-1 (0.0011 < p < 0.043). Also, tumours of stressed mice showed a tendency towards fewer total CD4 cells, more regulatory T cells (Treg), less T cell/tumour cell contacts and a reduction of CTLA-4 in CD4 cells (p > 0.05). TGF-beta in vitro was increased by 23.4% using catecholamines (p < 0.012) and in vivo employing chronic stress (p < 0.001). After 5 weeks tumour volumes were 130% (p = 0.0061) larger and median survival reduced by 13.5% (p = 0.0058). Tumours expressed more VEGF (p = 0.0334), had greater microvessel densities (p = 0.047), and an increased MMP-9 expression (p = 0.0456). Beta-catecholamines increased proliferation in tumour cells by 18% (p < 0.0001) and migration by 78% (p = 0.0348) whereas the beta-blocker propranolol reduced these effects by 25% (p < 0.0001) and 53% (p = 0.045), respectively. When stressed tumour-bearing animals were treated with propranolol tumour volumes were reduced by 69% (p = 0.0088) and survival improved by 14% (p < 0.0058).ConclusionsThe potential treatment with beta-blockers of patients with pancreatic cancer or other malignancies should be further evaluated as an adjuvant anti-neoplastic agent in clinical trials.     

Human colorectal tumour infiltrating lymphocytes express activation markers and the CD45RO molecule, showing a primed population of lymphocytes in the tumour area.

This study investigated the phenotype of freshly isolated human tumour infiltrating lymphocytes (TIL) from 14 patients with colorectal tumours, and compared them with lymphocytes derived from the lamina propria of the unaffected mucosa and with lymphocytes derived from peripheral blood of the same patients. It was found that TIL expressed the activation markers CD25 and HLA-DR to a higher extent than the peripheral blood lymphocytes (p = 0.01), and that both lamina propria lymphocytes and TIL preferentially expressed the CD45RO + phenotype, associated with memory cells, in contrast with peripheral blood lymphocytes [corrected]. Both lamina propria lymphocytes and TIL contained few natural killer (NK) cells (CD3-CD56+) compared with peripheral blood lymphocytes (p = 0.001), and this was reflected in the cytotoxicity assays. After 1 to 2 weeks in culture with interleukin-2 100 U/ml, lymphocytes from all three compartments had a high cytolytic activity against all targets tested, consistent with the lymphokine activated killer cell phenomenon. No increase in the number of NK cells was noted after culture, but 20-30% of the T cells now coexpressed the CD56 molecule. This was most prominent in the CD8+ subset, but lymphokine activated killer cell activity was found in both CD4+ and CD8+ subsets. Possible tumour escape mechanisms are discussed.     

T cell receptor-zeta and granzyme B expression in mononuclear cell infiltrates in normal colon mucosa and colon carcinoma.

BACKGROUND: Whereas the presence of a lymphoid infiltrate has been associated with a favourable prognosis in colorectal carcinoma, the proliferative and cytotoxic responses of freshly isolated tumour infiltrating lymphocytes are frequently impaired. In mice, tumour induced immune suppression has been associated with a decreased expression of the zeta-chain of the T cell receptor (TCR)-CD3 complex, and loss of mRNA for granzyme B. AIM: To compare the expression of TCR-zeta and granzyme B in lymphocytes infiltrating normal colonic mucosa and Duke's A and D colorectal carcinomas. SPECIMENS: Paraffin wax embedded normal (n = 10) and malignant colonic mucosa (seven Dukes's A, nine Dukes's D). METHOD: Immunohistochemistry. RESULTS: The numbers of TCR-zeta + lymphocytes decreased from normal mucosa to Dukes's D carcinomas. In contrast, granzyme B+ lymphocytes were more frequent in Dukes's A carcinomas than in normal mucosa, but disappeared from advanced stage tumours. Granzyme B expressing cells were mainly CD3- (natural killer/lymphokine activated killer cells) in normal mucosa, but CD3+ in tumours, indicating the presence of activated cytotoxic T lymphocytes. In vitro culture of tumour infiltrating lymphocytes rapidly restored the expression of both molecules. CONCLUSION: The frequency of TCR-zeta + and granzyme B+ lymphocytes is decreased in advanced stage colorectal carcinomas. The restoration of expression during in vitro stimulation suggests the presence of tumour derived suppressive factors in situ.     

Natural Killer Cells—A New Cytotoxic Mechanism Against Tumours?

Summary: Natural killer cells–a new cytotoxic mechanism against tumours? P. Hersey, Aust. N.Z. J. Med., 1979, 9, pp. 464–472. Lymphoid cells from the blood of most normal human subjects can kill a variety of cultured tumour cells in vitro. The cytotoxic activity of these cells (now referred to as natural killer cells) was initially detected during studies on known cytotoxic cells such as T cells, antibody dependent effector (K) cells and macrophages and was regarded as having no biological importance. Studies over the past few years have suggested, however, that natural killer cells may represent a major surveillance mechanism against tumours in vivo and may be the only cytotoxic mechanism against many tumours when those based on antigen recognition by the “conventional immune” cells fail. The nature of these cells and their cytotoxic mechanism has received considerable attention, but has so far not been clearly defined. A considerable body of evidence suggests they are of the T cell lineage but if so they are a sub-population of T cells that develop independently of the thymus. Recent studies suggest that interferon may play a major role in the induction of natural killer activity and this finding promises to be an important advance in our understanding of this cytotoxic mechanism. The level of natural killer cell activity against tumours in both animals and humans appears to be genetically determined and may be linked to genes of the major histocompatibility complex. Evidence for the biological importance of these cells was suggested by experimental studies where the in vivo susceptibility of animals to tumour growth could be related to the levels of their natural killer cell activity. Conversely, the take of different tumour cells in animals was inversely related to their susceptibility to natural killer cells. Studies on transplantation of bone marrow in animals also indicated that natural killer cell activity may be the predominant mechanism responsible for rejection of these grafts. Studies of a similar nature in humans are as yet limited but initial results from those on melanoma patients support the view that these cells are also important against tumours in human subjects. No information is yet available as to their importance in rejection of bone marrow grafts in human recipients. NK cells are therefore the latest of the effector cells to be defined after T cells, 6 cells, macrophages and K cells. These studies suggest that measurement of the activity of these cells warrants further attention in patients with tumours and bone marrow transplants and that methods to alter their activity may assume an important role in therapeutic procedures against tumours or in transplantation of bone marrow.     

Induction and clinical utilization of lymphokine-activated killer cells in patients with gastrointestinal tract cancers

In 18 patients with cancers of the gastrointestinal tract, lymphokine-activated killer (LAK) cell activity was studied and compared with that of healthy subjects. After cultivation with 10(3) iu/mL of recombinant interleukin-2, the cytotoxicity of patients' lymphoid cells was increased from 13.6 +/- 6.8% to 76.2 +/- 19.5% against Daudi cells and from 12.8 +/- 8.1% to 76.2 +/- 19.5% against K-562 cells. Based on these results, autologous LAK cells were given to patients. LAK cells injected into subdermal metastatic tumours demonstrated a significant inhibitory effect on tumour growth in comparison with that of control tumour nodules. Of four patients with metastatic tumours in the liver, to whom LAK cells were administered via the hepatic artery, tumour size was reduced by about 25% (minor response) in one patient, with a decrease of computerized tomography attenuation in the tumours occurring in the other three patients.     

Hepatocyte growth factor ameliorates the progression of experimental autoimmune myocarditis: a potential role for induction of T helper 2 cytokines

Hepatocyte growth factor (HGF) plays a role in cell protection, antiapoptosis, antifibrosis, and angiogenesis. However, the role of HGF in the immune system is not well defined. We examined the influence of HGF on T cells and the effects of HGF therapy in acute myocarditis. Lewis rats were immunized on day 0 with cardiac myosin to establish experimental autoimmune myocarditis (EAM). Human HGF gene with hemagglutinating virus of the Japan-envelope vector was injected directly into the myocardium on day 0 or on day 14 (two groups of treated rats). Rats were killed on day 21. Expression of c-Met/HGF receptor in splenocytes and myocardial infiltrating cells was confirmed by immunohistochemical staining or FACS analysis. Myocarditis-affected areas were smaller in the treated rats than in control rats. Cardiac function in the treated rats was markedly improved. An antigen-specific T cell proliferation assay was done with CD4-positive T cells isolated from control rats stimulated with cardiac myosin. HGF suppressed T cell proliferation and production of IFN-gamma and increased production of IL-4 and IL-10 secreted from CD4-positive T cells in vitro. Additionally, TUNEL assay revealed that HGF reduced apoptosis in cardiomyocytes. HGF reduced the severity of EAM by inducing T helper 2 cytokines and suppressing apoptosis of cardiomyocytes. HGF has potential as a new therapy for myocarditis.     

Testosterone, 11-Ketotestosterone, and Estradiol-17β Modify Baseline and Stress-Induced Interrenal and Corticotropic Activity in Trout

Estradiol-17β (E), 11-ketotestosterone (KT), and testosterone (T) were administered to immature rainbow and brown trout by implantation of steroid-containing cocoa butter pellets. This procedure elevated the levels of these hormones in the blood of the treated fish and had significant effects on plasma ACTH and cortisol levels in both unstressed and stressed rainbow trout and in stressed brown trout. E treatment significantly elevated resting levels of ACTH and cortisol and KT significantly suppressed resting ACTH levels in rainbow trout, although no effect of KT was noted on baseline cortisol levels. One hour of confinement stress increased ACTH levels in rainbow trout, but less so in T- and KT-implanted fish than in sham-implanted fish. A similar pattern was observed in stress-induced plasma cortisol levels where T and KT treatment of rainbow trout resulted in a more than 50% attenuation of plasma cortisol levels while E implantation significantly increased stress-induced plasma cortisol levels. In brown trout subjected to confinement stress for 96 hr, within 1 hr of the onset of confinement the stress-induced increase in plasma ACTH and plasma cortisol was significantly lower in T- and KT-implanted fish than in sham-implanted controls. However, these differences were not sustained at subsequent sample points during the 96-hr period of continuous confinement. Nonetheless, overall mean ACTH levels for the entire confinement period were significantly enhanced in E-implanted brown trout and significantly reduced in KT-implanted fish. Overall mean cortisol levels were significantly lower in T- and KT-implanted fish. The enhancement of stress responsiveness observed in E-treated immature fish was not observed during confinement stress in untreated mature female trout, with naturally high plasma E levels. However, untreated mature male trout displayed a significantly reduced cortisol response to confinement. It is suggested that gonadal steroids are involved in the regulation of both baseline and stress-induced activity of the pituitary–interrenal axis in salmonid fish.     

Chronic stress associated with spousal caregiving of patients with Alzheimer's dementia is associated with downregulation of B-lymphocyte GH mRNA

BACKGROUND: It has been demonstrated that growth hormone (GH) is synthesized and secreted by human peripheral mononuclear cells (PBMC), and the expression of GH mRNA can be found throughout the human immune system. METHODS: We studied a population of female caregivers of patients with Alzheimer's dementia (AD) who suffered from the stress of caring for these patients. We utilized quantitative RT-PCR to determine GH mRNA levels in T- and B-cell populations from PBMC. Subjects were nine caregivers of AD patients and nine age- and sex-matched controls. RESULTS: In the control group we found a threefold greater GH mRNA expression in B cells than in T cells. This finding was consistent with our previous in situ hybridization observation, suggesting GH mRNA in predominately B-cell areas of immune organs in humans. We also found that the expression of GH mRNA from total peripheral blood mononuclear cells and B cells in caregivers was 50% and 60% respectively less than that in the control group. CONCLUSIONS: Because the B-cell population is the source of antibody production, our findings suggest that the decrease in B-cell GH mRNA may contribute to the poor immune response to influenza virus vaccination that has been reported previously in chronically stressed caregivers.     

Lysis of primary hepatic tumours by lymphokine activated killer cells

Lymphokine activated killer cell is a newly described lytic system against a variety of solid tumours and is distinct in several respects from the classic cytolytic T cell and the natural killer systems. This study was conducted to evaluate the lytic activity of lymphokine activated killer cells against fresh autologous and allogeneic, as well as cultured hepatocellular carcinoma cells. Lymphokine activated killer cell was generated by incubating peripheral blood mononuclear cells with various concentrations of recombinant IL-2 (rIL-2, Cetus, USA) for various periods of time. A four hour 51Cr release assay was used to measure cytotoxicity. The results show that fresh and cultured hepatocellular carcinoma cells were only slightly susceptible to natural killer cells. Normal hepatocytes were resistant to lymphokine activated killer-mediated lysis. Lymphokine activated killer cells could be generated from mononuclear cells of hepatocellular carcinoma patients and normal subjects with lytic activity against fresh autologous and allogeneic and cultured hepatocellular carcinoma cells, but lymphokine activated killer cells from the former was less efficient than that from the latter. It is concluded that the adoptive immunotherapy with combined rIL-2 and lymphokine activated killer may be worth trying in early cases of primary hepatocellular carcinoma.     

Modulation of intestinal immune reactivity by interleukin 2

There is evidence indicating that interleukin 2 may be important in the regulation of intestinal immunity, as suggested by its capacity to induce nonspecific cytotoxic (lymphokine-activated killer) activity from human intestinal mucosal mononuclear cells. The present study was designed to further explore the phenotypic and functional changes induced by interleukin 2 on intestinal lymphocytes derived from inflammatory bowel disease and control tissues. Immunohistology of intestinal mucosa demonstrated few cells bearing the activation antigen recognized by anti-Tac (anti-interleukin 2 receptor) monoclonal antibody. However, when isolated lamina proprial mononuclear cells were exposed to interleukin 2 in culture, the number of Tac-positive cells increased dramatically, a phenomenon paralleled by the generation of lymphokine-activated killer cell activity. This cytotoxic function was critically dependent on the continuous availability of interleukin 2, but not on the expression of the Tac antigen, since Tac-negative cells were also cytotoxic. Depletion of natural killer cells, fractionation into T cell-enriched and -depleted cells before and after culturing with interleukin 2, and separation into Tac-positive and -negative cells after interleukin 2 activation failed to eliminate lymphokine-activated killer cell activity, suggesting that this phenomenon is mediated by phenotypically and functionally heterogeneous cell subsets. During the induction of lymphokine-activated killer cells variable amounts of interferon-gamma were produced, but these did not correlate with the degree of cytotoxicity. No differences were observed between the response to interleukin 2 by inflammatory bowel disease and control cells. Therefore, in view of its capacity to induce significant phenotypic and functional changes in different subpopulations of intestinal mucosal mononuclear cells, interleukin 2 should be regarded as an important modulator of intestinal immune reactivity.     

Isolation of intestinal mononuclear cells: factors released which affect lymphocyte viability and function.

Whether toxic or immunomodulatory factors are released during the collagenase digestion phase of the isolation of mononuclear cells from human intestinal mucosa was investigated by assessing the effect of the collagenase supernatant on the viability and natural killer activity of normal peripheral blood mononuclear cells. Three hours' incubation in collagenase supernatant suppressed natural killer activity by 25 +/- 4% and decreased the viability of peripheral blood mononuclear cells by 11 +/- 2%. The ability of collagenase supernatants to kill 51Cr-labelled peripheral blood mononuclear cells over four hours was assessed in 16 collagenase supernatants, eight of which produced lysis of 20 +/- 4%. There was no ultrastructural evidence of early degenerative changes in the viable intestinal mononuclear cells fresh from the isolation process or in peripheral blood mononuclear cells incubated in collagenase supernatant. Because prostaglandins are known to inhibit natural killer activity, PGE was measured in 20 collagenase supernatants by radioimmunoassay and found to be high at 27.5 +/- 4.0 ng/ml. Addition of indomethacin to the collagenase medium, however, failed to abolish the inhibitory effect of collagenase supernatant on natural killer activity and did not increase the natural killer activity of isolated intestinal mononuclear cells. The release of cytotoxic and immunomodulatory factors during the isolation of intestinal mononuclear cells indicates the necessity for careful assessment of the potential effects of the isolation process on any function being examined and casts doubt upon the relationship between in vitro findings and in vivo functional capabilities.     

Circadian variation in the sensitivity of the pituitary-adrenal system to dexamethasone suppression

Intact female rats were injected with 100 microgram dexamethasone/kg body weight or saline at 12.00, 24.00 or 04.00 h. Four hours later, i.e. 16.00, 04.00 or 08.00 h respectively, a double bleeding procedure was used to obtain blood samples from stressed and unstressed rats for subsequent fluorometric determination of corticosterone levels in the plasma. The difference between the level of corticosterone is stressed and unstressed rats (the stress increment) was determined and used as an index of the response of corticosterone in the plasma to stress. Increments in the levels of corticosterone in the plasma evoked by stress in rats injected at 12.00 h with dexamethasone and bled 4 h later were significantly (P less than 0.05) less than those in rats given dexamethasone at 24.00 or 04.00 and bled at 04.00 or 08.00 h respectively. In contrast, stress-induced increments in the level of corticosterone in the plasma of rats treated with saline did not vary with the time of day.     

Influence of Chronic Dietary Ethanol on Cytokine Production by Murine Splenocytes and Thymocytes

Prolonged consumption of ethanol (ETOH) results in alterations of host defense via immune modulation, increasing susceptibility to infection. In the present study, effects of chronic dietary ETOH on cytokine production by splenocytes and thymocytes, splenocyte and thymocyte proliferation induced by mitogens, splenic natural killer cell activity, and antibody production (IgA and IgG) were examined. C57BL/6 mice were fed 5% ETOH v/v in the Lieber-DeCarli liquid diet for 11 weeks. Release of interleukin (IL)-2, IL-5, IL-6, IL-10, and interferon (IFN)-γ produced by concanavalin A (Con A) stimulated splenocytes was significantly decreased, whereas secretion of IL-4 was slightly decreased by chronic dietary ETOH compared with controls. Production of tumor necrosis factor-α and IL-6 by lipopolysaccharide-stimulated splenocytes was significantly and slightly decreased by ETOH compared with controls, respectively. Splenocyte and thymocyte proliferation induced by Con A was significantly inhibited by ETOH, whereas splenocyte proliferation induced by lipopolysaccharide was not affected. Natural killer cell activity was significantly inhibited by ETOH compared with controls. The production of IgA and IgG by splenocytes were also significantly decreased by ETOH compared with controls. The levels of IL-2, IL-4, and IL-6 produced by Con A-stimulated thymocytes were significantly reduced by dietary ETOH compared with control, whereas production of IFN-γ by thymocytes was not affected. Our results suggest that chronic dietary ETOH alters the cytokine release, thereby impairing immune response and T-cell maturation, which increase host susceptibility to infection.     

Effects of dietary iron overload on progression in chemical hepatocarcinogenesis.

AIM: The present study was undertaken to investigate possible effects of dietary iron during the progression step in hepatocarcinogenesis. METHODS: Two experiments were performed, in which preneoplastic foci were produced in rat liver using the Solt & Farber protocol, with diethylnitrosamine as initiator and partial hepatectomy + 2-acetylaminofluorene as promoter. Two weeks after promotion, animals were fed 1.25-2.5% dietary carbonyl iron or a control diet until sacrifice. In the first experiment, animals were killed at different time points when they developed an abdominal mass in combination with weight loss. In the second experiment, animals were sacrificed 45 weeks post-promotion. Liver tumours were counted and histologically graded. Tumour levels of ubiquinone-9 and alpha-tocopherol were determined with HPLC, and labelling and apoptotic indices calculated using immunohistochemistry. The number and area of glutathione S-transferase 7,7 (GST-7,7)-positive foci were determined. RESULTS: In experiment number 1, survival and tumour differentiation were similar in iron-treated animals and controls. In the second experiment, iron-treated rats had an increased number of GST-7,7-positive foci compared to controls. Number and size of carcinomas were similar between the groups, whereas tumour differentiation was higher in rats exposed to iron. Cell proliferation, apoptosis and concentrations of alpha-tocopherol in tumours were not altered by iron. The ratio of reduced/oxidized ubiquinone-9 was decreased in tumours from iron-treated animals. CONCLUSION: In this model, dietary iron overload resulted in an increased number of preneoplastic foci but did not enhance the progression of these into hepatocellular carcinomas. Iron decreased the ratio of reduced/oxidized ubiquinone-9 in tumours, indicating that neoplastic liver cells utilize intracellular ubiquinones as a defense mechanism against iron-induced oxidative stress.     

Lymphokine-activated killer cell activity in patients with primary and metastatic malignant liver tumors

Lymphokine-activated killer cells were generated from peripheral blood mononuclear cells of 33 patients with liver tumors (benign, 6; primary malignant, 10; metastatic, 17) and 10 healthy individuals. Although peripheral blood mononuclear cell yield was significantly lower (p less than 0.01) in patients with hepatocellular carcinoma or with metastatic colorectal cancer, natural killer activity in the peripheral blood mononuclear cell fraction was comparable in all groups tested. Optimal lymphokine-activated killer activity was demonstrated after 9 to 12 days of culture in recombinant interleukin 2. Lymphokine-activated killer activity, interleukin 2-induced lymphocyte proliferation and total lytic activity generated per culture in all patient groups studied were similar to those of normal control cells cultured under the same conditions. These in vitro data demonstrate the feasibility of obtaining lymphokine-activated killer cells from the blood of patients with liver tumors and provide a rationale for the future use of lymphokine-activated killer cells in adoptive immunotherapy of patients with primary and metastatic hepatic neoplasms.     

Location of superoxide anion generation in human colonic mucosa obtained by biopsy.

To identify which cells generate superoxide, inflamed human mucosa was tested with nitro-blue tetrazolium as a probe, because it is reduced by strong reducing agents to form insoluble blue formazan, which then precipitates in tissues. Biopsy specimens from control subjects and patients with ulcerative colitis were studied. The specimens were organ cultured with bubbling air or nitrogen, and inhibition of the reduction by catalase (a hydrogen peroxide scavenger), para-benzoquinone (a tissue permeable superoxide scavenger), or superoxide dismutase (a superoxide scavenger) was assayed. The dye was reduced by epithelial cells, vascular endothelium, and infiltrating mononuclear cells of the mucosa. Its reduction by vascular endothelium and infiltrating mononuclear cells was greater in inflamed mucosa. The reduction by vascular endothelium and infiltrating mononuclear cells was inhibited in cultures with nitrogen saturation or with 1 mM para-benzoquinone. The vascular endothelium seems to produce superoxide in the inflamed mucosa, which would exacerbate tissue injury in ulcerative colitis.     

FOXP3 positive regulatory T-cells in cutaneous and systemic CD30 positive T-cell lymphoproliferations

The CD30-positive lymphoproliferations encompass a spectrum of disorders that share histological and phenotypic similarities but differ markedly in clinical behaviour. The basis for this diversity is not known, but it has been proposed that immune suppression by cytokines and/or regulatory T-cells (Tregs) may be implicated. In this study, skin biopsies from lymphomatoid papulosis (LyP) (n = 14), primary cutaneous anaplastic large cells lymphoma (C-ALCL) (n = 13) and systemic anaplastic large cells lymphoma (S-ALCL) with (n = 9) or without (n = 6) ALK expression were examined by immunohistology for FOXP3 expression in tumour cells and tumour infiltrating Tregs. Labelling of a majority of the neoplastic cells was seen in one case of C-ALCL. Another three cases (one LyP and two C-ALCL) displayed weak labelling of very occasional atypical T-cells. In the remaining 38 cases the atypical lymphoid infiltrate was FOXP3 negative. By contrast, all biopsies contained tumour infiltrating FOXP3-positive Tregs. Significant higher numbers were recorded in ALK negative S-ALCL and LyP than in C-ALCL and S-ALCL positive for ALK. In conclusion, it is shown that FOXP3 expression in cutaneous and systemic CD30-positive lymphoproliferations is generally confined to tumour infiltrating Tregs. These cells may have influence upon the clinical behaviour, possibly depending upon the net degree of Treg mediated immune suppression of tumour cells relative to tumour infiltrating, cytotoxic effector cells, thereby implicating the more favourable outcome of LyP compared to C-ALCL.